CN118086361A - Application of gene BcWRKY1 in regulation and control of yellow-green phenotype of black vegetable leaves - Google Patents

Abstract

The application provides an application of a gene BcWRKY1 in regulating and controlling the yellow-green phenotype of black vegetable leaves, and relates to the technical field of genetic engineering, wherein the nucleotide sequence of the gene BcWRKY1 is shown as SEQ ID NO.4, and the expression of a gene BcCLH2 is promoted by inhibiting the expression of the gene BcWRKY, so that the black vegetable leaves turn yellow from green; by promoting the expression of the gene BcWRKY and inhibiting the expression of the gene BcCLH2, the vegetable leaves can be promoted to turn from yellow to green; the nucleotide sequence of the gene BcCLH is shown as SEQ ID NO.1; the gene provided by the application is beneficial to genetic breeding of Wucai.

Description

Application of gene BcWRKY1 in regulation and control of yellow-green phenotype of black vegetable leaves

Technical Field

The invention relates to the technical field of genetic engineering, in particular to application of a gene BcWRKY1 in regulating and controlling yellow-green phenotype of black vegetable leaves.

Background

Wu Cai (Brassica campesttris L. Ssp. Chinese (L.) Makino var. Rosularis TSEN ET LEE) is a subspecies of Chinese cabbage (B.campestris L.), which is native to China and is mainly distributed in the river basin of Jiang Huai. It is a nutritious vegetable rich in minerals and vitamins and exhibits a five-colored six-colored leaf. The yellow black vegetable variety W7-2 has green inner leaf at normal temperature in the plant period and is turned yellow gradually after low temperature. The black vegetable after turning into yellow is soft and juicy in mouth and delicious in taste.

Therefore, research on the molecular mechanism of the black vegetable turning into yellow provides a new thought or theoretical basis for culturing the black vegetable yellowing variety, and is a problem to be solved urgently.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides application of a gene BcWRKY1 in regulating and controlling the yellow-green phenotype of the black vegetable leaves, so as to solve the problems of undefined molecular mechanism of black vegetable turning in the prior art and the like.

In order to achieve the above purpose, the invention is realized by the following technical scheme:

In a first aspect of the invention, an application of a gene BcWRKY1 in regulating and controlling the yellow-green phenotype of black vegetable leaves is provided, and a nucleotide sequence of the gene BcWRKY1 is shown as SEQ ID NO. 4.

Further, the application includes:

by inhibiting the expression of gene BcWRKY, the expression of gene BcCLH2 is promoted, the leaf of Wu Cai is changed from green to yellow, or

By promoting the expression of the gene BcWRKY, the expression of the gene BcCLH is inhibited, and the vegetable leaves are promoted to turn from yellow to green;

The nucleotide sequence of the gene BcCLH is shown as SEQ ID NO.1.

Further, the expression of the repressor gene BcWRKY includes knocking out the gene BcWRKY1.

Further, the expression of the facilitator gene BcWRKY1 comprises constructing an over-expression vector for the gene BcWRKY 1.

Further, the Wucai variety is W7-2 or WS-1.

In a second aspect of the present invention, there is provided a method of modulating chlorophyllase activity in leaf of wu-chia, the method comprising:

promoting expression of the gene BcCLH2 by inhibiting expression of the gene BcWRKY, promoting promotion of chlorophyllase activity in leaf of Wucai, or

By promoting the expression of the gene BcWRKY, the expression of the gene BcCLH2 is inhibited, and the activity of chlorophyllase in the leaf of the Wucai is reduced.

In a third aspect of the present invention, there is also provided the use of gene BcWRKY, a protein encoded by gene BcWRKY1 or a biological material containing gene BcWRKY1 for the preparation of a product for promoting the conversion of black-vegetable leaves from green to yellow or from yellow to green; the nucleotide sequence of the gene BcWRKY is shown as SEQ ID NO. 4.

Further, the biological material is a plasmid, an expression vector or a transgenic cell.

In a fourth aspect, the invention also provides an application of the gene BcCLH2 in regulating and controlling the yellow-green phenotype of the black vegetable leaves.

Further, the application includes:

promoting the expression of gene BcCLH and promoting the leaf of Wucai to turn yellow from green; or (b)

Inhibiting the expression of gene BcCLH and promoting the vegetable leaves to turn from yellow to green.

The application of the gene BcWRKY1 in regulating and controlling the yellow-green phenotype of the black vegetable leaves has the following beneficial effects compared with the prior art:

The provided gene BcWRKY is related to the regulation of the yellow-green phenotype of the black vegetable leaves, and can be used for controlling the yellow-green phenotype of the black vegetable leaves by inhibiting or over-expressing the gene BcWRKY, so that the cultivation of a new variety of the black vegetable with the target leaf phenotype is facilitated, and a new idea is provided for genetic breeding.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is an agarose gel electrophoresis of gene BcCLH;

FIG. 2 is a comparison of RT-qPCR analysis and chlorophyllase activity assay of gene BcCLH2 at three periods;

FIG. 3 is a graph of transient expression analysis and subcellular localization of tobacco of gene BcCLH;

FIG. 4 is a diagram showing transient expression analysis of the Wucai gene BcCLH;

FIG. 5 is a plot of self-activated concentration screens for pAbAi-BcCLH 2;

FIG. 6 is a graph showing the results of pAbAi-BcCLH2 screening in a yeast cDNA library of Wucai;

FIG. 7 is a diagram showing the interaction of gene BcWRKY and gene BcCLH2 in a yeast single hybridization experiment;

FIG. 8 is a cross-plot of the dual luciferase reporter assay verifying gene BcWRKY and gene BcCLH 2.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In order to solve the problem that the yellow turning mechanism of the Wucai is not clear in the prior art, the main idea of the application is as follows:

the gene BcWRKY1 related to the phenotype of the Wu Cai She Huanglu is provided, and through up-regulating or inhibiting the expression of the gene, the change of the Wu Cai leaves from green to yellow or from yellow to green can be effectively regulated, so that a new idea is provided for genetic breeding of the Wu Cai.

The present application provides gene BcWRKY1 for functional verification by the following examples.

Example 1

In this example, the Wucai W7-2 variety was selected as the material (W7-2 was gradually subjected to low temperature in autumn and winter during growth and leaves turned yellow).

The following operations are performed on the Wucai W7-2:

Planting W7-2 seedling in greenhouse of 26+ -2deg.C (daytime) and 20+ -2deg.C (night), with illumination intensity of 300 μmol m ^-2s^-1 and relative humidity of 70-80%; in this process, the black-vegetable leaves remain green (the corresponding state of the black-vegetable leaves obtained in this process is before turning yellow (G)).

Transplanting seedlings growing to 7-8 leaves into a low-temperature growth chamber with 6+/-2 ℃ (daytime) and-1+/-2 ℃ (nighttime), 300 mu mol m ^-2s^-1 light intensity and 70-80% relative humidity, and culturing for 12d; in the process, the black vegetable leaves are gradually changed from green to yellow (the corresponding state of the black vegetable leaves obtained in the process is changed into yellow (Y)).

Then, setting the temperature to 26+/-2 ℃ (daytime) and 20+/-2 ℃ (night), the illumination intensity to 300 mu mol m ^-2s^-1, and the relative humidity to 70-80%, and continuing for 12d; in the process, the black vegetable leaves are gradually changed from yellow to green (the corresponding state of the black vegetable leaves obtained in the process is changed into green (RG)).

In the above procedure, three fully developed young leaves were collected every 3 days from the center.

1. The method for extracting total RNA and synthesizing cDNA of plant leaves in three periods of before (G), after (Y) and after (RG) turning yellow comprises the following specific steps:

RNA from samples of three periods was extracted with the extraction TAKARA RNA kit, and after quantification, the RNA was reverse transcribed with the TRAN reverse transcription kit, with the system shown in Table 1.

TABLE 1

2. Cloning, recovering and purifying of Wucai gene BcCLH2

The nucleotide sequence of gene BcCLH (shown as SEQ ID NO. 1) is obtained in the Wu-CAI genome, specifically:

The homology arm primer (F: SEQ ID NO.5; R: SEQ ID NO. 6) was designed by CE-Design software based on the gene sequence and pCAMBIA2300 vector sequence, and was sent to the general company for synthesis of the primer sequence. The sample was applied according to the system of Table 2, and the PCR was performed at 95℃for 5min,95℃for 30s,60℃for 30s,72℃for 2min, and 72℃for 5min. After the reaction, the PCR products were identified by 1% agarose gel electrophoresis (FIG. 1). The recovery of the target DNA fragment (nucleotide sequence shown as SEQ ID NO. 1) was performed according to TIANGEN agarose gel DNA recovery kit.

TABLE 2

3. Connection carrier and transformed escherichia coli

Adding sample according to the system of Table 3, sucking and beating, mixing, connecting at 37deg.C for 30min, and rapidly placing on ice to obtain connection product (containing pCAMBIA2300-BcCLH and pCAMBIA 2300). Adding 10 mu L of the ligation product into 100 mu L of escherichia coli, gently sucking and beating, uniformly mixing, standing for 5min on ice, performing heat shock at 42 ℃ for 45-60 s, rapidly transferring to ice, and standing for 2min. Then 700 mu L of LB without antibiotics is added into the centrifuge tube, the mixture is mixed uniformly and resuscitated at 37 ℃ for 20min at 200rpm, 100 mu L of resuscitated liquid is evenly coated on a culture medium containing 100mg/mL Kan antibiotics, and then the culture medium is placed into an incubator for inversion culture at 37 ℃ for overnight. 8 single colonies were picked the next day in 500. Mu.L LB centrifuge tubes of 100mg/mL Kan, and shaken on a shaker at 37℃for 5h until the bacterial solution became turbid, to obtain bacterial solution.

TABLE 3 Table 3

Taking the bacterial liquid obtained in the last step, and sampling by using a gene upstream primer F, SEQ ID NO.5 and a vector downstream primer R, wherein the SEQ ID NO.7 is according to a system of a table 4, and the PCR procedures are 95 ℃ for 5min,95 ℃ for 30s,60 ℃ for 30s,72 ℃ for 2min and 72 ℃ for 5min. After the reaction, the PCR products are subjected to 1% agarose gel electrophoresis identification, and then are sent to a general company for sequencing, if the comparison is successful, the genes are successfully connected with the carrier, bacterial liquid and plasmids are returned, the bacterial liquid and the glycerol are mixed according to the ratio of 1:1, quick frozen by liquid nitrogen, and the products are stored at the temperature of minus 80 ℃.

TABLE 4 Table 4

4. Agrobacterium transformation

100. Mu.L of Agrobacterium are taken and competent, then 5. Mu.L of the obtained ligation product (pCAMBIA 2300-BcCLH 2) is added, the mixture is sucked and stirred uniformly, and placed on ice for 5min, liquid nitrogen for 5min, water bath at 37 ℃ for 5min, 700. Mu.L of LB without antibiotics is added, and shaking is carried out for 3h at 28 ℃. After harvesting at 6000rpm for one minute, 100. Mu.L of the supernatant was left and resuspended in pellet form by gentle pipetting and plating on LB plate containing 100mg/mL Kan (kanamycin) and 25mg/mL rif (rifampicin) and inverted for 2 days in a 28 degree incubator, 8 individual colonies were picked up in 500. Mu.L LB centrifuge tubes of 100mg/mL Kan and 25mg/mL rif and shaken at 28℃for 24h until the bacterial solution became turbid. The PCR procedure was carried out at 95℃for 5min,95℃for 30s,60℃for 30s,72℃for 2min and 72℃for 5min, according to the system of Table 4. After the reaction, the PCR product is subjected to 1% agarose gel electrophoresis to identify that the PCR product is qualified, the PCR product is mixed with glycerol according to the ratio of bacterial liquid to glycerol of 1:1, quick frozen by liquid nitrogen, and stored at the temperature of minus 80 ℃; obtaining agrobacterium transformation.

Example 2

1. Determination of the expression level of Wucai Gene BcCLH2 in different periods

The expression level of gene BcCLH in three stages of G, Y and RG is measured by the following specific method:

According to the nucleotide sequence of the gene BcCLH shown in SEQ ID NO.1, designing a fluorescent primer (BcCLH-F: SEQ ID NO.8; bcCLH2-R: SEQ ID NO. 9) of the gene BcCLH2 by using Oligo7 software for detecting the expression level of the gene BcCLH2 and taking the Wucaiaction as an internal reference gene, wherein the primer is F: SEQ ID NO.10; r is SEQ ID NO.11, and the primer sequence is synthesized by general biological company. The qRT-PCR system is as follows: GREEN QPCR Supermix 10. Mu.L, bcCLH-F0.4. Mu.L, bcCLH 2-R0.4. Mu.L, cDNA 1. Mu.L, DEPC 8.2. Mu.L. The qRT-PCR procedure was: 95 ℃ 30s,95 ℃ 5s,60 ℃ 15s,72 ℃ 10s, image acquisition, 40 times of circulation, 58 ℃ to 95 ℃ 5s of dissolution curve, and image acquisition.

2. Chlorophyll enzyme Activity assay

0.1G of the sample was weighed, 1mL of PBS was added thereto, and the mixture was ground with a grinder. After sufficient grinding, centrifugation is carried out at 2000rpm for 20 minutes, and the supernatant is aspirated into a 1.5ml centrifuge tube. Setting a standard substance hole and a sample hole, wherein 50 mu L of standard substances with different concentrations are added into the standard substance hole; adding 40 mu L of sample diluent into a sample hole to be detected on an ELISA plate, then adding 10 mu L of sample to be detected, and gently shaking and uniformly mixing; 100 mu L of enzyme-labeled reagent is added into each hole except for blank plates; incubating for 60 minutes at 37 ℃ after membrane sealing by a sealing plate; diluting the 20 times concentrated solution with distilled water 20 times for use. Carefully removing the sealing plate film, discarding the liquid, spin-drying, filling the washing liquid in each hole, standing for 30s, discarding, repeating for 5 times, and beating; adding 50 mu L of a color developing agent A and 50 mu L of a color developing agent B into each hole, gently oscillating and uniformly mixing, and developing for 15min at 37 ℃ in a dark place; adding 50 mu L of stop solution into each hole to stop the reaction; the absorbance of each well was measured by adjusting the blank well to 0 and the wavelength of 450nm, and the measurement was performed 15min after the addition of the stop solution.

As shown in FIG. 2A, the qRT-PCR analysis result shows that the expression level of the gene BcCLH is obviously up-regulated from the G period to the Y period, and the expression level is obviously down-regulated from the Y period to the RG period, and as shown in FIG. 2B, the activity of the chlorophyllase is obviously increased from the G period to the Y period, and the activity is reduced from the Y period to the RG period.

3. Transient expression analysis and subcellular localization of tobacco of gene BcCLH2

Activation buffer preparation: MES (2- (N-morpholino) ethanesulfonic acid) and MgCl ₂ in a ratio of 5:1, and sterilizing at high temperature. The Agrobacterium transformants obtained in example 1 were shaken overnight in a shaker at 28℃until the OD of the bacterial solution reached 0.8-1.0, centrifuged at 4000rpm for 8min, the supernatant removed, suspended with 3ml of ddH ₂ O, centrifuged at 4000rpm for 8min, suspended with Activation buffer, and centrifuged at 4000rpm for 8min. By using Activation buffer as a control, the OD value of the bacterial liquid is measured, and Activation buffer is used for adjusting the OD value of the bacterial liquid to 0.8-1.0. The bacterial liquid is as follows: as=1000: 1, AS (acetosyringone) is added into the bacterial liquid. After stationary culture at 28℃for 3 hours, nicotiana benthamiana was injected. After injection, the cells were subjected to dark treatment for 1 day, and after incubation under light for about 2 days, the positioning results were observed by confocal laser. In addition, leaf phenotype after tobacco injection was observed, leaf color difference and total chlorophyll content were measured; the method comprises the following steps:

(1) Color difference value measurement

Determination of color value: leaf colorimetric values were measured on the upper surface of the leaf every 3 days after planting using a colorimeter (DS-700D), 10 plants were measured, and the measurement was repeated 3 times at the same leaf positions. And (5) performing color data analysis by adopting a CIELAB color coordinate system. Wherein, the color difference value has three representative color values: l, a, b. L represents brightness (black and white), a represents red-green, and b represents yellow-blue. In addition, there are two chromatic aberrations: value H (Hue angle) and value C (Chroma).

(2) Chlorophyll content determination

The content of photosynthetic pigments (Chl a, chl b) was measured by the Arnon method: fresh leaves were ground to a powder, then 2.5ml of 80% acetone and a small amount of calcium carbonate and quartz sand were added and ground to a homogenate. 3ml of acetone was added thereto and the mixture was left to stand in the dark for 3 to 5 seconds. A piece of filter paper was placed in a funnel, moistened with ethanol, the extract was poured into the funnel along a glass rod and filtered into a 25ml volumetric flask. Sucking 95% ethanol with a dropper, cleaning the mortar, filtering the cleaning solution into a volumetric flask, eluting pigment with 95% ethanol along the periphery of the filter paper, and finally fixing the volume to scale with 95% ethanol after the filter paper and residues are completely whitened. Absorbance at 646 and 663nm wavelengths was measured using a TU1950 ultraviolet visible spectrophotometer (PER-SEE, beijing, china). Wherein, the content of Chl a (mg/g) and Chl b (mg/g) is calculated as follows:

CChl a mg/L＝12.21×A663-2.81×A646；

CChl b mg/L＝20.13×A646-5.03×A663；

Chl a mg/g＝CChl a mg/L×V(L)/Mfresh；

Chl b mg/g＝CChl b mg/L×V(L)/Mfresh。

As shown in the test result in FIG. 3, through transient expression of gene BcCLH2 in Nicotiana benthamiana, the result shows that compared with a control, the Nicotiana benthamiana over-expressed gene BcCLH2 has a chlorosis phenomenon (FIG. 3A), and the Nicotiana benthamiana can be turned from green to yellow by over-expressing gene BcCLH; the color difference values and total chlorophyll content are shown in table 5, and compared with the control, the L Value was up-regulated by 8.07%, the B Value was up-regulated by 18.07%, the a Value was not significantly changed, the total Chl content was down-regulated by 52.6%, and the subcellular localization result showed that the gene BcCLH was localized in chloroplasts (fig. 3B).

From the above, the over-expression of gene BcCLH can promote the conversion of the Nicotiana benthamiana from green to yellow.

TABLE 5

4. Analysis of the transient expression of Wucai from Gene BcCLH2

Leaf blade 'WS-1' of evergreen variety of Wucai is injected according to the method of transient expression of tobacco, leaf blade phenotype after injection is observed, and leaf blade color difference value and total chlorophyll content are measured.

As a result of transiently expressing gene BcCLH of Wu-Cai, the leaf of Wu-Cai over-expressed gene BcCLH showed yellowing compared with the control, as shown in FIG. 4. The color difference values and total chlorophyll content are shown in table 6, and the L Value, a Value and b Value are all significantly up-regulated, and the total Chl content is significantly down-regulated, compared to the control. These results indicate that gene BcCLH2 plays a key role in chlorophyll degradation of the leaf in wu-chia (i.e., overexpression of gene BcCLH2 results in turning wu-chia leaves from green to yellow).

TABLE 6

Example 3

1. Gene BcCLH2 Yeast library screening

Extracting promoter sequence (nucleotide sequence shown as SEQ ID NO. 2) of gene BcCLH from Wu Cai gene, selecting-1800 to-1000 fragment (nucleotide sequence shown as SEQ ID NO. 3) to construct bait carrier for library screening according to cis-acting element analysis, comprising:

Construction of pAbAi-BcCLH2 recombinant plasmid according to the procedure in example 1, wherein the primer sequence is F: SEQ ID NO.12; r is SEQ ID NO.13; after construction, 1ug pAbAi-BcCLH recombinant plasmid was linearized using BbsI restriction enzyme and the specific cleavage system is shown in Table 7:

TABLE 7

After one hour of digestion, the mixture was recovered. According to the system of Table 8, sucking and beating, mixing, water-bath at 30deg.C for 30min (turning over for 6-8 times for mixing at 15 min), water-bath at 42deg.C for 15min (turning over for 6-8 times for mixing at 7.5 min). Centrifuging at 5000rpm for 40s, removing supernatant, re-suspending dd H ₂ O400 mu L, centrifuging for 30s, removing supernatant, removing dd H ₂ O50 mu L, coating SD/-Ura plates, culturing for 3-5 days, and selecting 4-5 single clones on the SD/-Ura plates for colony PCR identification; wherein, the CARRIER DNA is also subjected to the following pretreatment before the sample is added: it was inserted into a metal bath at 95℃for 5min, heated and rapidly inserted onto ice. After agarose gel electrophoresis, it was confirmed whether the size of the inserted band was normal. Selection of the correct monoclonal for AbA concentration screening. The identification primer is pAbAi-F, SEQ ID NO.14; pAbAi-R SEQ ID NO.15.

TABLE 8

Bait carrier pAbAi-BcCLH2 was selected and cultured in 50ml YPDA (adenine sulfate yeast extract peptone glucose medium) at 30℃at 230-250 rpm until OD600 reached 0.4-0.5. Then, Y1H Gold yeast cells were collected and resuspended with 3ml of 1.1 XPEG/LiAc. Mu.g of yeast library plasmid (yeast library was constructed by Shanghai European biomedical sciences Co., ltd.) and 50. Mu. LCARRIER DNA were added and mixed. Then adding 1 XPEG/LiAc 2.5ml, and culturing in water bath at 30deg.C for 45min; adding 160 mu LDMSO (dimethyl sulfoxide), and culturing in water bath at 42deg.C for 20min; YPD (Yeast extract peptone glucose medium) 3ml was added and incubated at 30℃for 90min. Finally, the cells were suspended in 1ml of 0.9% NaCl solution. The cultured bacteria were diluted 10-fold, 100-fold, 1000-fold, 10000-fold and incubated at 30℃for 3-5 days in SD/-leu plates and SD/-leu/150. Mu.L AbA plates, respectively. Clone identification was performed when the single clone grew to 1-2 mm. The results were as follows:

Recombinant plasmid pAbAi-BcCLH2 can be grown on SD/-Ura plates, indicating that recombinant plasmid can successfully transform yeast cells and 150ng/ml AbA can inhibit the growth of background bacteria (FIG. 5). Y1HGold (pAbAi-BcCLH 2) was screened in a yeast library. A total of 33 clones were screened on SD/-Leu/AbA (150 ng/ml) screens (FIG. 6). Gene BcWRKY1 (transcription factor, corresponding nucleotide sequence as shown in SEQ ID NO. 4) was selected by positive clone sequencing.

3. Yeast single hybridization and double luciferase reporter experiments verify that gene BcWRKY and gene BcCLH interact

(1) Yeast Single hybridization experiment

The prey vector pGADT7-BcWRKY1 was constructed according to the procedure of example 1, primer sequence F: SEQ ID NO.16; r is SEQ ID NO.17. Bait carrier pAbAi-BcCLH2 was selected and cultured in 50ml YPDA (adenine sulfate yeast extract peptone glucose medium) at 30℃at 230-250 rpm until OD600 reached 0.4-0.5. Then, pAbAi-BcCLH2 yeast cells were collected and resuspended with 3ml of 1.1 XPEG/LiAc. Mu.g of pGADT 7-BcWRKY/pGADT 7 recombinant plasmid and 5. Mu. LCARRIER DNA were added and mixed. Then adding 1 XPEG/LiAc 2.5ml, and culturing in water bath at 30deg.C for 30min; adding 20 mu LDMSO, and culturing in water bath at 42 ℃ for 15min; centrifuging at high speed for 15s, and discarding the supernatant. Finally, the cells were suspended in 100. Mu.L of 0.9% NaCl solution. The cultured bacteria were diluted 10-fold, 100-fold, 1000-fold, 10000-fold and incubated on SD/-leu plates and SD/-leu/150. Mu LAbA plates, respectively, for 3-5 days at 30 ℃.

As shown in FIG. 7, pAbAi-BcCLH2+pGADT7 and pAbAi-BcCLH2+pGADT7-BcWRKY1 were able to grow on SD/-leu plates, demonstrating that pGADT7-BcWRKY1 and pGADT7 were successfully transformed into Y1H Gold yeast cells; pAbAi-BcCLH2+pGADT7-BcWRKY1 white colonies grew on SD/-leu/150. Mu. LAbA plates, pAbAi-BcCLH2+pGADT7 did not grew, demonstrating that gene BcWRKY1 could interact with gene BcCLH 2.

(2) Double luciferase reporter assay

LUC-BcCLH2 recombinant plasmid and pCAMBIA2300-BcWRKY1 were constructed according to the procedure in example 1, the primers were respectively of sequence LUC-BcCLH2-F: SEQ ID NO.18, LUC-BcCLH-R: SEQ ID NO.19 and pCAMBIA2300-BcWRKY1-F: SEQ ID NO.20, pCAMBIA2300-BcWRKY1-R: SEQ ID NO.21. LUC-BcCLH O, pCAMBIA2300-BcWRKY1 and pCAMBIA2300 agrobacterium transformants are respectively shaken overnight in a shaking table at 28 ℃, the OD value of the bacterial liquid is preferably 0.8-1.0, the bacterial liquid is centrifuged at 4000rpm for 8min, the supernatant is removed, 3ml of ddH ₂ O is firstly used for suspension, the suspension is further used for suspension at 4000rpm for 8min, and the suspension is further used for suspension at Activation buffer and the centrifugation at 4000rpm for 8min, so that three bacterial liquids are obtained. And taking Activation buffer as a control, measuring the OD value of each bacterial liquid, and regulating the OD value of each bacterial liquid to be 0.8-1.0 by using Activation buffer to obtain three target bacterial liquids. 1, the method comprises the following steps: 1, LUC-BcCLH2 is corresponding to the target bacterial liquid: mixing the target bacterial solutions corresponding to pCAMBIA2300-BcWRKY1 and the target bacterial solution corresponding to LUC-BcCLH2 of a control group: the pCAMBIA2300 agrobacterium transformation corresponds to the target bacterial liquid in a ratio of 1:1, mixing in proportion to obtain two mixed bacterial solutions; finally, mixing bacterial liquid: as=1000: 1, AS (acetosyringone) was added to each mixed bacterial liquid. After stationary culture at 28℃for 3 hours, the same leaves of Nicotiana benthamiana were simultaneously injected at different positions. After injection, the cells were dark-treated for 1 day, and after incubation under light for about 2 days, the luminescence was observed in a living body imager (see FIG. 8).

We determined how gene BcWRKY1 regulated the activity level of gene BcCLH2 by a dual luciferase assay. The results showed that gene BcWRKY negatively regulated the expression level of gene BcCLH2 (fig. 8).

In conclusion, the application of the gene BcWRKY1 in regulating and controlling the yellow-green phenotype of the black vegetable leaves can be used for genetic breeding of the black vegetable, and a new idea is provided for cultivation of related varieties.

It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. The application of the gene BcWRKY1 in regulating and controlling the yellow-green phenotype of the black vegetable leaves is provided, and the nucleotide sequence of the gene BcWRKY1 is shown as SEQ ID NO. 4.

2. The application of claim 1, wherein the application comprises:

by inhibiting the expression of gene BcWRKY, the expression of gene BcCLH2 is promoted, the leaf of Wu Cai is changed from green to yellow, or

By promoting the expression of the gene BcWRKY, the expression of the gene BcCLH is inhibited, and the vegetable leaves are promoted to turn from yellow to green;

The nucleotide sequence of the gene BcCLH is shown as SEQ ID NO.1.

3. The use of claim 2, wherein inhibiting expression of gene BcWRKY comprises knocking out gene BcWRKY1.

4. The use of claim 2, wherein said promoting expression of gene BcWRKY comprises constructing an overexpression vector for gene BcWRKY 1.

5. The use according to claim 1, wherein the Wucai variety is W7-2 or WS-1.

6. A method of modulating chlorophyllase activity in a leaf of wu-sedge, the method comprising:

promoting expression of the gene BcCLH of claim 1 by inhibiting expression of the gene BcWRKY of claim 1, promoting promotion of chlorophyllase activity in leaf of Wucai, or

By promoting the expression of the gene BcWRKY of claim 1, the expression of the gene BcCLH of claim 1 is inhibited, and the activity of chlorophyllase in the leaf of wu is reduced.

7. Use of gene BcWRKY1, the protein encoded by gene BcWRKY1, or a biological material comprising gene BcWRKY1 for the preparation of a product for promoting the conversion of black vegetable leaves from green to yellow or from yellow to green; the nucleotide sequence of the gene BcWRKY is shown as SEQ ID NO. 4.

8. The use according to claim 7, wherein the biological material is a plasmid, an expression vector or a transgenic cell.

9. Use of the gene BcCLH of claim 2 for regulating the yellow-green phenotype of wu-chia-pee leaves.

10. The application of claim 9, wherein the application comprises:

promoting the expression of gene BcCLH and promoting the leaf of Wucai to turn yellow from green; or (b)

Inhibiting the expression of gene BcCLH and promoting the vegetable leaves to turn from yellow to green.